The human body has several barriers that protect its integrity and shield it from mechanical, chemical, and microbial harm. The various barriers include the skin, intestinal and respiratory epithelia, blood–brain barrier (BBB), and immune system. In the present review, the focus is on the physical barriers that are formed by cell layers. The barrier function is influenced by the molecular microenvironment of the cells forming the barriers. The integrity of the barrier cell layers is maintained by the intricate balance of protein expression that is partly regulated by microRNAs (miRNAs) both in the intracellular space and the extracellular microenvironment. The detection of changes in miRNA patterns has become a major focus of diagnostic, prognostic, and disease progression, as well as therapy-response, markers using a great variety of detection systems in recent years. In the present review, we highlight the importance of liquid biopsies in assessing barrier integrity and challenges in differential miRNA detection.

(1) Background: Tuberous sclerosis complex (TSC) mutations directly affect mTORC activity and, as a result, protein synthesis. In several cancer types, TSC mutation is part of the driver mutation panel. TSC mutations have been associated with mitochondrial dysfunction, tolerance to reactive oxygen species due to increased thioredoxin reductase (TrxR) enzyme activity, tolerance to endoplasmic reticulum (ER) stress, and apoptosis. The FDA-approved drug rapamycin is frequently used in clinical applications to inhibit protein synthesis in cancers. Recently, TrxR inhibitor auranofin has also been involved in clinical trials to investigate the anticancer efficacy of the combination treatment with rapamycin. We aimed to investigate the molecular background of the efficacy of such drug combinations in treating neoplasia modulated by TSC mutations. (2) Methods: TSC2 mutant and TSC2 wild-type (WT) cell lines were exposed to rapamycin and auranofin in either mono- or combination treatment. Mitochondrial membrane potential, TrxR enzyme activity, stress protein array, mRNA and protein levels were investigated via cell proliferation assay, electron microscopy, etc. (3) Results: Auranofin and rapamycin normalized mitochondrial membrane potential and reduced proliferation capacity of TSC2 mutant cells. Database analysis identified peroxiredoxin 5 (Prdx5) as the joint target of auranofin and rapamycin. The auranofin and the combination of the two drugs reduced Prdx5 levels. The combination treatment increased the expression of heat shock protein 70, a cellular ER stress marker. (4) Conclusions: After extensive analyses, Prdx5 was identified as a shared target of the two drugs. The decreased Prdx5 protein level and the inhibition of both TrxR and mTOR by rapamycin and auranofin in the combination treatment made ER stress-induced cell death possible in TSC2 mutant cells.

Background: The anabolic window hypothesis suggests a limited post-exercise period for optimal nutrient uptake and utilization. Prior research indicates that miRNAs in extracellular vesicles (EVs) may regulate post-exercise adaptation by influencing protein synthesis. This study aimed to examine the effects of resistance exercise (RE) on physiological parameters and the expression and function of miRNAs transported in EVs. Methods: Twenty resistance-trained male participants (22 ± 2 years) completed a five-week RE program designed for hypertrophy. They consumed maltodextrin and whey protein based on assigned nutrient timing: immediately post-exercise (AE), three hours post-exercise (AE3), or no intake (CTRL). Body composition and knee extensor strength were assessed. Small EVs were isolated and then validated via three methods. Nanoparticle tracking analysis determined EV concentration and size, followed by pooled miRNA profiling and signaling pathway analysis. Results: Skeletal muscle mass significantly increased in AE (p = 0.001, g = 2) and AE3 (p = 0.028, g = 1), and it was higher in AE compared to CTRL (p = 0.013, η² = 0.41), while knee extensor strength improved only in AE (p = 0.032, g = 0.9). Body fat percentage significantly decreased in all groups, AE (p = 0.005, g = 1.5), AE3 (p = 0.024, g = 1), and CTRL (p = 0.005, g = 1.7). Vesicle concentration significantly increased in the AE group (p = 0.043, r = 0.7), while it decreased in the CTRL group (p = 0.046, r = 0.8). Distinct miRNA expression profiles emerged post-intervention: 20 miRNAs were upregulated in AE, while 13 in AE3 and 15 in CTRL were downregulated. Conclusions: Nutrient timing influences training adaptation but is not more critical than total macronutrient intake. Changes in EV-transported miRNAs may regulate anabolic processes via the PI3K-AKT-mTOR and FoxO pathways through PTEN regulation.

Background: Although regular physical activity is known to reduce cancer risk, the circulating molecular mechanisms responsible for these effects remain incompletely defined. Exercise alters the release and composition of extracellular vesicles (EVs), suggesting a role in systemic anti-tumor signaling. However, the contribution of large EVs (L-EVs) ─ a distinct and understudied EV subtype ─ to the regulation of lung adenocarcinoma cell behaviour has not been examined to date. Methods: L-EVs were isolated from platelet-free plasma of healthy, regularly exercising older adults with over 25 years of training history (aged 61 ± 2 years), collected at rest (Pre) and after (Post) an acute endurance exercise bout. EV fractions were characterized via transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and antibody-based profiling. PC9 lung adenocarcinoma cells were incubated with plasma-derived L-EV–enriched fractions for 24 h. Cell viability and metabolic activity were evaluated using ATP-based luminescence and XTT assays following L-EV treatment. miRNA and gene expression changes were assessed using RT-qPCR with TaqMan arrays. Differentially expressed miRNAs (DEmiRs) and genes (DEGs) were analysed using Ingenuity Pathway Analysis (IPA) to evaluate regulatory networks and predict functional outcomes. Results: Exercise significantly increased plasma nanoparticle concentration (mean increase of 5.05 × 10⁷ ± 9.99 × 10⁶ particles/mL; p < 0.001, Cohen’s d = 1.685). TEM and NTA confirmed successful isolation of L-EVs, with mode sizes of 136.1 ± 23.6 nm pre-exercise and 129.5 ± 17.7 nm post-exercise. In PC9 cells, both Pre- and Post-L-EVs reduced metabolic activity compared to control conditions, as demonstrated by ATP-based and XTT assays, indicating a functional impact on cell viability ( p < 0.001). Transcriptomic analysis revealed distinct expression changes between conditions. Post-L-EVs downregulated oncogenic and immune evasion–associated genes (MAX, FASLG, and ELK1) while upregulating pro-apoptotic and cell cycle inhibitory genes (CASP9, FADD, and CDKN2B). Several DEmiRs, including miR-21-5p, miR-301b-3p, and miR-193a-3p, shifted in directions opposite to their known oncogenic patterns in NSCLC, suggesting a tumor-suppressive influence. IPA predicted enhanced apoptotic signaling and reduced metastatic potential following Post-L-EV treatment. Conclusion: Exercise-conditioned plasma-derived enriched L-EVs modulate gene and miRNA expression in lung adenocarcinoma cells toward a tumor-suppressive profile. By attenuating oncogenic and immune-evasion signals while enhancing apoptotic and cell cycle–inhibitory pathways, Post-L-EVs offer preliminary mechanistic insight into the anti-cancer potential of physical activity. Their origin from older adults engaged in long-term exercise highlights the systemic benefits of sustained activity and the promise of L-EVs as biomarkers of exercise responsiveness.